Formulations and methods for treating hypercoagulable states

ABSTRACT

A method of treating a human patient with a hypercoagulable state or protein C deficiency, which comprises administering activated Protein C and protein C Zymogen.

This is the national phase application, under 35 USC 371, forPCT/US01/11771, filed 14 May 2001, which claims the priority of U.S.provisional application No. 60/206,733, filed 24 May 2000.

This invention relates to methods for treating vascular occlusivedisorders and hypercoagulable states, including sepsis, disseminatedintravascular coagulation, purpura fulminans, major trauma, surgery,burns, adult respiratory distress syndrome (ARDS), transplantations,deep vein thrombosis, heparin-induced thrombocytopenia, sickle celldisease, thalassemia, viral hemorrhagic fever, thromboticthrombocytopenic purpura, hemolytic uremic syndrome, acute coronarysyndromes (ACS; e.g. unstable angina, myocardial infarction) and proteinC deficiency characterized by administering a combination of activatedProtein C (aPC or APC) and protein C zymogen (PCZ). The invention alsoincludes inventive formulations comprising aPC, PCZ and apharmaceutically acceptable carrier.

Protein C is a serine protease and naturally occurring anti-coagulantproduced as an inactive precursor or zymogen by the liver. Human proteinC is made in vivo as a single polypeptide of 461 amino acids. Thispolypeptide undergoes multiple post-translational modificationsincluding: 1) cleavage of a 42 amino acid signal sequence; 2) cleavageof lysine and arginine residues (positions 156 and 157) to make a2-chain inactive zymogen (a 155 amino acid residue light chain attachedvia a disulfide bridge to a 262 amino acid residue heavy chain); 3)vitamin K-dependent carboxylation of nine glutamic acid residues locatedwithin the amino-terminal 45 residues (gla-domain); and, 4) carbohydrateattachment at four sites (one in the light chain and three in the heavychain). The protein C zymogen circulates in the plasma and, upon removalof a dodecapeptide at the N-terminus of the heavy chain, results in aPCpossessing enzymatic activity. aPC plays a key role in regulatinghemostasis by inactivating Factors V_(a) and VIII_(a) in the coagulationcascade.

Blood coagulation is a highly complex process regulated by the balancebetween pro-coagulant, anti-coagulant, and fibrinolytic mechanisms. Thisbalance determines a condition of either normal hemostasis or abnormalpathological thrombus generation and the progression of hypercoagulablestates. Two major factors control this balance; the generation of fibrinand the activation and subsequent aggregation of platelets, bothprocesses controlled by the generation of the enzyme thrombin, whichoccurs following activation of the clotting cascade. Thrombin, incomplex with thrombomodulin, also functions as a potent anti-coagulantsince it activates protein C zymogen to the active enzyme, aPC. In largeblood vessels, the activation of protein C zymogen to aPC bythrombin-thrombomodulin complex is further augmented by an endothelialtransmembrane protein, endothelial-protein receptor (EPCR)[Stearn-Kurosawa, et al. Proc. Natl. Acad. Sci. USA 93:10212–10216,1996]. APC, in turn inhibits the generation of thrombin. Thus, throughthe feedback regulation of thrombin generation via the inactivation ofFactors Va and VIIIa, aPC functions as perhaps the most importantdown-regulator of blood coagulation resulting in protection againstthrombosis.

Additionally, aPC exerts profibrinolytic properties that facilitate clotlysis and exerts anti-inflammatory effects via inhibiting the release ofinflammatory mediators, such as, tumor necrosis factor and variousinterleukins.

Recombinant aPC has completed Phase III clinical trials for treatingsevere sepsis. In addition, Phase III studies directed to the use ofplasma derived human aPC in DIC, where severe sepsis is a subset of thepatients studied, have been completed in Japan. The use of aPC to treatcongenital and acquired protein C deficiency and to treat disseminatedintravascular coagulation (DIC) and hypercoagulopathy have also beenreported. Wada, et al., 1993, Am. J. Hematol. 44:218–219; Sugimoto, etal., 1997, Thromb. Haemost. 77:1223–1224; Oh, et al., 1998, Blood,92:4402; Aoki, et al., 1998, J. New Remedies & Clinics 47:448–482).

Several encouraging pre-clinical studies using aPC in animal models havealso been reported. A study in a baboon sepsis model by Taylor, et al.,[J. Clin. Invest. 79:918–25, 1987], used plasma-derived human aPC. Fiveout of five animals survived 7 days and were considered permanentsurvivors to the experimental protocol. In control animals receiving anidentical infusion of E. coli, five out of five animals died in 24 to 32hours. The use of recombinant aPC for treating thrombotic occlusion orthromboembolism in a baboon model was also disclosed by Griffin, et al.in U.S. Pat. No. 5,084,274 and European Patent Application EP 0 318 201B1.

In a lipopolysaccharide (LPS; E. coli) sepsis model in rats [Murakami,et al., Blood 87:642–647, 1996], the pulmonary vascular injury inducedby LPS was inhibited by human plasma-derived aPC. Furthermore, in aligation and puncture sepsis model in rabbits, Okamoto, et al.,[Gastroenterology 106:A747, 1994], demonstrated that plasma-derivedhuman aPC was effective in protecting the animals from coagulopathy andorgan failure.

Plasma derived PCZ is approved for treating congenital protein Cdeficiency in both Europe and Japan. The use of human PCZ to treatcongenital and acquired protein C deficient states has also beenreported. See: (e.g. Dreyfus, et al., Semin. Thromb. Hemost. 21:371–381,1995; Minford, et al., Br. J. Haematol., 93:215–216, 1996; Smith, etal., Lancet, 350:1590–1593, 1997). In addition, plasma-derived human PCZhas been used as a successful adjunct to aggressive conventional therapyin the management of forty-seven patients with purpura fulminans inbacterial sepsis of which forty survived (Gerson, et al., Pediatrics91:418–422, 1993; Smith, et al., Lancet 350:1590–1593, 1997; White, etal., Blood 92:670a #2765, 1998; Ettingshausen, et al., Semin. Thromb.Hemost. 25:537–541, 1999; Betrosian, et al., Crit. Care Med.27:2849–2850, 1999; Rintala, et al., Lancet 347:1767, 1996; Rivard, etal., J. Pediatr. 126:646–652, 1995). Gerson, et al., [1993] describetreatment of a child with proven gram positive bacteremia and purpurafulminans, who was failing to respond to aggressive conventionaltreatment. The patient was treated with plasma-derived human PCZresulting in an associated correction of coagulopathy and DIC, andarrest of clinical signs of the development of septic shock-relatedpurpura fulminans.

Rintala, et al., [1996] reported the treatment of 2 adults withmeningococcal septicemia presented with purpura fulminans. The patientswere treated with plasma-derived PCZ at 400 μg/kg bolus every six hoursfor 8–10 days. One died and one survived. Rivard, et al., [1995]reported the treatment of four patients with meningococcemia and purpurafulminans, who all survived following human PCZ therapy. Smith, et al.,[1997] and White, et al., [1998] treated thirty patients withmeningococcemia with plasma-derived PCZ at about 400 μg/kg bolusfollowed by 60 μg/kg/hour infusion for several days. Twenty-seven of thethirty patients survived. Ettingshausen, et al., [1999] treated eightmeningococcemia patients with plasma-derived PCZ at a bolus of about320–480 μg/kg followed by about 200 μg/kg up to once every 4 hours.Median treatment time was three days and the range was from one tosixteen days. Six of the eight patients survived. Betrosian, et al.,[1999] treated one patient with meningococcemia with plasma-derived PCZat 400 μg/kg every 6 hours the first day and 240 μg/kg every six hoursthe subsequent two days. The patient did not survive.

Plasma-derived human PCZ has also been reported to successfully treatother hypercoagulable or acquired protein C deficient states such asveno-occlusive disease as a complication of bone marrow transplantation(Veldman, et al., Bone Marrow Transplant. 21:S238, 1998), devastatingcoagulopathy of unknown etiology (Favier, et al., Hematol. Cell Therapy40:67–70, 1998), and heparin-induced thrombocytopenia (Boshkov, et al.,Blood 94:102b, 1999).

PCZ and aPC differ structurally and also differ pharmacokinetically andpharmacodynamically. PCZ is a pharmaceutically inactive protein and inhumans is present in concentrations of about 4 μg/ml. PCZ is convertedto aPC by thrombin in complex with an endothelial membrane protein,thrombomodulin in the microvascular beds. In larger blood vessels, theconversion of PCZ to aPC is further facilitated by EPCR in complex withthrombin-thrombomodulin. Therefore, aPC prolongs aPTT clotting time intreated subjects while PCZ does not. APC also circulates in humans at amuch lower concentration of about 2 ng/ml. APC has a half-life in humansof about 23–45 minutes, more than 10 fold shorter than the PCZ(approximately 10 hours). Shock, Vol. 12, No. 3, 243–244, 1999. Onereason for the short half-life is that blood levels of aPC are regulatedby molecules known as serpins (Serine Protease Inhibitors), whichcovalently bind to aPC forming an inactive serpin/aPC complex. Theserpin/aPC complexes are formed when aPC binds and proteolyticallycleaves a reactive site loop within the serpin; upon cleavage, theserpin undergoes a conformational change irreversibly inactivating aPC.The serpin/aPC complex is then eliminated from the bloodstream primarilyvia hepatic receptors for the serpin/aPC complex.

A major lesson from various studies is that early intervention intreating hypercoagulable states, such as sepsis, is more likely tosucceed. Evidence is suggesting that the earlier the therapeuticintervention relative to the inflammatory challenge, the more likelythat a drug will have a beneficial effect. Dellinger, et al., Chest,1997, 111:744–53. Toshiaki, et al., J Am Coll Surg, 1998, 187: 321–329.(Early diagnosis is now considered critical in severe sepsis . . .Because treatment has poor results when begun in the later stages ofsepsis, treatment should be started as soon as possible).

Although early therapeutic intervention is preferred, for a variety ofmedical reason (e.g., potential adverse effects and drug interactions),it is often undesirable to treat a patient with aPC prior toconfirmation of a hypercoagulable or protein C deficient state. Inaddition, disadvantages of treatment with PCZ alone also exist. Forexample, it takes time to convert the inactive PCZ to the active anduseful aPC. Furthermore, some patients may be unable, or have a reducedcapacity, to convert PCZ to aPC. For example, certain cytokines releasedinto the circulation during hypercoagulable states may down-regulate theamount of thrombomodulin on endothelial cell surfaces and thus, impedethe rate of protein C activation (Moore, et al., Blood 73:159–165,1989). Faust, et al., also report that the molecular machinery needed toconvert PCZ to aPC is down-regulated in septic patients. Shock 2000: 13(Suppl.):29(abst.#12). Moreover, aPC may be more resistant to neutrophilelastase than PCZ (Philapitsch, et al., Thromb. Haemost, 69:A664, 1993).Elastase is elevated in sepsis (Setiz, et al., Eur. J. Haematol.43:22–28, 1989; Jansen, et al., Blood 87:2337–2344, 1996). Therefore,administration of aPC alone and administration of PCZ alone may resultin less than optimal treatment of hypercoagulable states and/or proteinC deficient states.

The present invention is the first to describe the combination of aPCand PCZ in the treatment of hypercoagulable states and/or protein Cdeficiency. The combination of aPC and PCZ results in a treatment thatallows for the reduction of the dosages of both aPC and PCZ and animprovement of clinical treatment options and outcome of the patientbeing treated. Reducing the amount of an agent in this combinationtherapy may result in reduced side effects that may occur with eitheragent, particularly with the potential for bleeding that may be inducedby administering a higher dose of aPC. This combination is particularlypreferred for those patients prone to bleeding episodes or at high riskof bleeding, for example, due to inherited bleeding disorders and/or forpatients taking therapeutic agents that may increase the risk ofbleeding episodes. This combination also allows for a more rapidtreatment approach (PCZ) followed by administration of aPC uponconfirmation of a protein C deficient state and/or a hypercoagulablestate or when a clinician suspects a patient may be unable to convertPCZ to the active form of the protein, aPC.

The combination therapy of PCZ and aPC may be administered in anysequence or combination according to the best combination for aparticular patient or disease need. The present invention is intended toencompass all dosing regimens employing aPC and PCZ for preventing ortreating a condition disclosed herein. The needs of a particular patientand the preferences of a treating physician may result in aPC and PCZbeing used in a variety of dosing schedules. For example, PCZ may beadministered prior to aPC. APC may be administered prior to PCZ. PCZ andaPC may also be administered simultaneously at different combinationproportions. PCZ and aPC may also be administered with varying doses andalternating back and forth between the two agents. The following areexamples of possible dosing options and are not intended to limit thescope of the invention in any way. PCZ and aPC are preferablyadministered parenterally, for example, by an intravenous orsubcutaneous route. PCZ and aPC may also be administered in bolus,loading dose, or continuous infusion.

PCZ administered before aPC: for patients that are at high risk ofdeveloping sepsis, for example, PCZ may be given prophylactically toprevent the patients from developing sepsis or severe sepsis. Asexplained in more detail below, patients at greater risk of developingsepsis include patients with cancer who are undergoing chemotherapy;patients undergoing other trauma or surgeries, such as, abdominalsurgeries or an organ transplantation; severely burned patients; andpregnant patients. For example, it is known that protein C levels candecrease to 40% of normal level in neutropenic patients (cancer patientsthat were treated with intense chemotherapy) some 12 hours before theonset of clinical symptoms of septic shock. Mesters, et al., Crit. CareMed., in press). The low level of protein C in this group of patientswas predictive of the mortality outcome of the patients. This group ofpatients that showed this dramatic drop in protein C all died. Thosethat did not have this drop of protein C survived. So the physiciansthat are caring for patients that are at high risk of developing sepsiscan monitor the level of protein C. As soon as protein C starts todecrease below a normal level, the physician can administer PCZ to thepatient. PCZ can be given subcutaneously (for example, at a dose of(about 200–2000 μg/kg) once a day to once every 2–3 days depending onthe protein C consumption rate to maintain the endogenous PCZ level atabout 100% of normal or intravenously at a dose of about 200–700 μg/kgonce a day. This prophylactic treatment with PCZ may reduce thedevelopment and/or severity of severe sepsis. APC is then administeredto treat the patient upon development of sepsis, severe sepsis, orseptic shock.

APC during the acute phase of severe sepsis and septic shock isconsidered better treatment than PCZ because, as previously stated,thrombomodulin, which is required to convert PCZ to aPC in the patientmay be down-regulated due to inflammation during sepsis (Moore, et al.1989). Also, PCZ is more susceptible to degradation by neutrophilelastase than aPC (Philapitsch, et al., Thromb. Haemost, 69:A664, 1993).Circulating neutrophil elastase is elevated during severe sepsis.

After treating the patient with aPC for about 1 to about 10 days oruntil the patient's endogenous protein C level is near the normal level,the physician may again administer PCZ (and continue or discontinue theaPC) for a period of time until all signs of coagulopathy are resolved.Patients who may need this last stage of PCZ treatment may be patientswho developed purpura fulminans during the severe sepsis/septic shockstage. There may be necrotic tissues or digits that may need skingrafting or amputation. The ischemic tissues that are beyond salvage maycontinue to induce low-grade coagulopathy until the proper proceduresare performed. Other patients who may benefit from the above-describeddosage regimen include patients who suffer from trauma, burns orpatients undergoing or who are about to undergo organ transplantationand those patients developing veno-occlusive disease.

aPC given before PCZ: for a patient presenting to the hospital orphysician with obvious symptoms of a hypercoagulable state, for example,severe sepsis or septic shock, these patients may be treated immediatelywith aPC for about 1 to about 10 days or until the patient's endogenousprotein C level is above the lower limit of the normal range (the lowerlimit of normal is about 60–80%, as described in more detail herein). Anexample of this scenario is a patient presenting with purpura fulminansfrom either gram negative or gram positive bacteremia, or viral orparasitic infection. This patient will be treated with aPC first, forexample, at a dose of about 10 μg/kg/hr to about 36 μg/kg/hr for about 1to about 10 days followed by treatment with PCZ at a dose of (200–600μg/kg once a day) for an additional period of 1 to about 7 days, up toseveral weeks. An additional example is directed to treating a patientwho presents with an acute phase of melioidosis. The patient will betreated with aPC, for example, at a dose of about 10 μg/kg/hr to about36 μg/kg/hr for 1 to about 10 days (with or without an initial aPCbolus). In that the pathogen that causes melioidosis, B. pseudomallei,requires 2 weeks to 6 months of anti-infective treatment for totaldisease remission, B. pseudomallei may be actively infecting the patientfor up to 6 months. This may result in a continuous or “chronic”acquired protein C deficiency requiring treatment with PCZ for up to 6months. The actual doses of aPC and PCZ may be varied to ultimatelyachieve activated Protein C plasma levels in the range of 2 ng/ml to 200ng/ml, preferably in the range of 10 ng/ml to 90 ng/ml. Another exampleis directed to treating a patient that is first diagnosed withcongenital protein C deficiency presenting with acute purpura fulminansor a patient with congenital protein C deficiency suffering from anacute crisis of thrombosis/hypercoagulopathy. This patient will betreated with aPC first, for example, at a dose of about 10 μg/kg/hr toabout 36 μg/kg/hr for about 1 to about 10 days followed by treatmentwith PCZ at a dose of (200–600 μg/kg once a day) for an additionalperiod of 1 to about 7 days, up to several weeks. One or two days intoPCZ therapy, chronic anti-coagulant therapy can be initiated while thePCZ therapy can be gradually weaned. Example of chronic anti-coagulanttherapy can be oral wafarin therapy, or heparin therapy (low-molecularweight heparin or unfractionated heparin given intravenously orsubcutaneously). These patients generally require chronic anti-coagulanttherapy for life. PCZ and aPC can be life-saving for this group ofpatients during acute thrombotic crisis.

Monitoring PCZ and aPC may be conducted by various methods, including anenzyme capture immunogenic method for measuring aPC levels; othermethodologies that are in development for measuring PCZ levels such as acombination of antigenicity and mass spectrometric method; andpreferably by commercially and clinically approved diagnostic kits forPCZ measurements using antigenicity or activity methodologies.

aPC and PCZ given simultaneously: In the above two scenarios, it may bedifficult to determine precisely when to transition from administeringPCZ to administering aPC or vice versa. So during the transition, thephysician may use aPC and PCZ together before transition into PCZ or aPCtherapy alone. In addition, a physician may decide to reduce the dose ofeither aPC or PCZ for a variety of reasons. A patient may be at anincreased risk of bleeding with aPC and lowering the aPC dose whilecontinuously administering PCZ may benefit the patient's hypercoagulablestate and/or protein C deficiency. A physician may want to administer alower dose of aPC, for example, 5 μg/kg/hr to about 20 μg/kg/hrsimultaneously with, for example, 20–100 μg/kg of PCZ by bolus whiledetermining whether the patient has the ability to convert PCZ to aPC invivo.

Examples of certain hypercoagulable states contemplated within the scopeof this invention are described below.

Sepsis

Sepsis is defined clinically as a systemic response to infection orsuspected infection complicated by one or more organ failures, sepsis isassociated with and mediated by the activation of a number of hostdefense mechanisms including the cytokine network, leukocytes, and thecomplement and coagulation/fibrinolysis systems. [Mesters, et al., Blood88:881–886, 1996]. Disseminated intravascular coagulation [DIC], withwidespread deposition of fibrin in the microvasculature of variousorgans, is an early manifestation of sepsis/septic shock. DIC is animportant mediator in the development of the multiple organ failuresyndrome and contributes to the poor prognosis of patients with severesepsis. [Fourrier, et al., Chest 101:816–823, 1992].

Blocking DIC has also been proposed as a new target for clinical trialsin sepsis [e.g., Levi, et al., JAMA 270:975–979, 1993]. However, simplyblocking the coagulation defect in sepsis may not be sufficient. Asreviewed by Esmon, [Arteriosclerosis & Thromb. 12:135–145, 1992],several antithrombotics have not shown efficacy in the baboon sepsismodel, including active site-blocked factor Xa [Taylor, et al., Blood78:364–368, 1991], hirudin and hirulog [Maraganore, Perspective in DrugDiscovery and Design 1:461–478, 1994]. Each of these antithromboticswere able to block the consumptive coagulopathy in the animals but werenot able to improve survival. Additionally, investigators in Japan[patent application JP7097335A] have proposed treating coagulopathyassociated with hepatic insufficiency, which has the potential ofdeveloping DIC-like symptoms, with plasma derived activated Protein C.

Purpura fulminans (ecchymotic skin lesions, fever, hypotensionassociated with bacterial sepsis, viral, bacterial or protozoaninfections) and/or DIC have been associated with numerous bacterial,viral, or protozoan infections which include but are not limited toinfections caused by Rickettsia (Rocky Mountain Spotted fever, tick bitefever, typhus, etc.) [Graybill, et al., Southern Medical Journal,66(4):410–413, 1973; Loubser, et al., Annals of Tropical Pediatrics13:277–280, 1993]; Salmonella (typhoid fever, rat bite fever) [Koul, etal., Acta Haematol, 93:13–19, 1995]; Pneumococci [Carpenter, et al.,Scand J Infect Dis, 29:479–483, 1997] Yersina pestis (Bubonic plague)[Butler, et al., The Journal of Infectious Disease, 129:578–584, 1974];Legionella pneumophila (Legionnaires Disease); Plasmodium falciparum(cerebral malaria) [Lercari, et al., Journal of Clinical Apheresis,7:93–96, 1992]; Burkholderia pseudomallei (Melioidosis); Pseudomonaspseudomallei (Melioidosis) [Puthucheary, et al., Transactions of theRoyal Society of Tropical Medicine and Hygiene, 86:683–685, 1992];Streptococci (Odontogenic infections) [Ota, Y., J. Japanese Assoc.Infect. Dis., 68:157–161]; zoster virus [Nguyen, et al., Eur J Pediatr,153:646–649, 1994]; Bacillus anthracis (Anthrax) [Franz, et al., Journalof the American Medical Assoc., 278(5):399–411, 1997]; Leptospirainterrogans (leptospirosis) [Hill, et al., Seminars in RespiratoryInfections, 12(1):44–49, 1997]; Staphylococci [Levin, M., PediatricNephrology, 8:223–229]; Haemophilus aegyptius (Brazilian purpuricfever); Neisseria (gonococcemia, meningococcemia); and mycobacteriumtuberculosis (miliary tuberculosis).

Even though the purpura fulminans, DIC or acquired protein C deficiencyconditions in sepsis/septic shock or other infections have been welldocumented as indicated above, there is no suggestion to treat thesepatients according to the inventive treatment methods of the presentapplication.

Transplantation

A variety of transplantation associated thromboembolic complications mayoccur following bone marrow transplantation (BMT), liver, kidney, orother organ transplantations [Haire, et al., JAMA 274:1289–1295, (1995);Harper, et al., Lancet 924–927 (1988); and Sorensen, et al., J. Inter.Med 226:101–105 (1989); Gordon, et al., Bone Marrow Transplan. 11:61–65,(1993)]. Decreased levels of circulating protein C have been reportedafter BMT [Bazarbachi, et al., Nouv Rev Fr Hematol 35:135–140 (1993);Gordon, et al., Bone Marrow Trans. 11:61–65 (1993)], renaltransplantation [Sorensen, et al., J. Inter. Med 226:101–105 (1989)],and liver transplantation [Harper, et al., Lancet 924–927(1988)]. Thisdeficiency in protein C contributes to a hypercoagulable state placingpatients at risk for thromboembolic complications.

For example, hepatic venocclusive disease (VOD) of the liver is themajor dose-limiting complication of pre-transplantation regimens forBMT. VOD is presumably the result of small intrahepatic venuleobliteration due to intravascular deposition of fibrin. [Faioni, et al.,Blood 81:3458–3462 (1993)]. In addition, VOD causes considerablemorbidity and mortality following BMT [Collins, et al., Throm. andHaemo. 72:28–33 (1994)]. A decreased level of protein C coincident withthe peak incidence of VOD has been reported [Harper, et al., Bone MarrowTrans. 5:39–42 (1990)] and is likely to be a contributing factor to thegenesis of this condition.

Organ dysfunction after BMT including pulmonary, central nervous system,hepatic or renal, is a complication that occurs in a high percentage oftransplant patients [Haire, et al., JAMA 274:1289–1295, (1995)]. Asingle organ dysfunction in BMT is a strong predictor of multiple organdysfunction syndrome (MODS) which is the leading cause of death in BMTpatients. Disseminated intravascular coagulation (DIC) due to a massiveactivation of the coagulation system and widespread deposition of fibrinin the microvasculature of various organs is an important mediator inthe development of MODS [Fourrier, et al., Chest 101:816–823 (1992)].Thus, a deficiency in protein C levels in patients who have undergonebone marrow or other organ transplantations leads to a hypercoagulablestate that predisposes the patients to venous thromboemboliccomplications and organ dysfunction. A need currently exists todetermine a method of treating humans with a hypercoagulable stateassociated with organ transplantations utilizing activated Protein C.

Burns

It has long been recognized that severely burned patients havecomplications associated with hypercoagulation [Curreri, et al., Ann.Surg. 181:161–163 (1974)]. Burned patients have supranormal in vitroclotting activity and frequently develop DIC which is characterized bythe sudden onset of diffuse hemorrhage; the consumption of fibrinogen,platelets, and Factor VIII activity; intravascular hemolysis; secondaryfibrinolysis; and biopsy evidence of microthrombi [McManis, et al., J.of Trauma 13:416–422, (1973)]. Recently, it was reported that the levelsof protein C were reduced drastically in severely burned patients andthat this reduction of the natural anticoagulant may lead to an increasein the risk of DIC [Lo, et al., Burns 20:186–187 (1994)]. In addition,Ueyama, et al., in discussing the pathogenesis of DIC in the early stageof burn injury, concluded that massive thrombin generation and decreaseof anticoagulant activity may occur in proportion to the severity ofburns [Ueyama, et al., Nippon Geka Gakkai Zasshi 92:907–12 (1991)]. DICis one of the common complications in patients suffering from severeburn injuries.

Protein C deficiency has been documented in severely burned patients asindicated above, however, there has never been a suggestion to treatpatients according to the methods disclosed in the present application.

Pregnancy

It is well known that pregnancy causes multiple changes in thecoagulation system which may lead to a hypercoagulable state. Forexample, during pregnancy and postpartum, the risk of venous thrombosisis almost fivefold higher than in the non-pregnant state. In addition,clotting factors increase, natural inhibitors of coagulation decrease,changes occur in the fibrinolytic system, venous stasis increases, aswell as increases in vascular injury at delivery from placentalseparation, cesarean section, or infection [Barbour, et al., ObstetGynecol 86:621–633, 1995].

Although the risk of a complication due to this hypercoagulable state inwomen without any risk factors is small, women with a history ofthromboembolic events are at an increased risk for recurrence when theybecome pregnant. In addition, women with underlying hypercoagulablestates, including the recent discovery of hereditary resistance to aPC,also have a higher recurrence risk [Dahlback, Blood 85:607–614, 1995].

Therefore, it has been suggested that women with a history of venousthromboembolic events who are found to have a deficiency inantithrombin-III, protein C, or protein S, are at an appreciable risk ofrecurrent thrombosis and should be considered for prophylacticanti-coagulant therapy [Conrad, et al., Throm Haemost 63:319–320, 1990].The conditions of preeclampsia and eclampsia and other obstetricalcomplications such as amniotic fluid embolism and placenta abruption inpregnant women appear to be a state of increased coagulopathy anddisseminated intravascular coagulation as indicated by an increase infibrin formation, activation of the fibrinolytic system, plateletactivation and a decrease in platelet count [Clin Obstet Gynecol35:338–350, 1992]. Preeclampsia is thought to be the result ofuteroplacental ischemia due to an anomaly of the “vascular insertion” ofthe placenta. Consequences of preeclampsia include hypertension as wellas DIC which leads to the release of numerous microthrombi which causeplacental, renal, hepatic and cerebral lesions [Rev Fr Gynecol Obstet86:158–163, 1991]. Furthermore, preeclampsia can lead to a severe andlife threatening condition known as the HELLP syndrome which is definedas preeclampsia complicated by thrombocytopenia, hemolysis and disturbedliver function [Rathgeber, et al., Anasth Intensivther Notfallmed25:206–211, 1990]. Additionally, it has been documented that there is areduction in protein C levels in pregnant women with severe preeclampsiawhen compared to normal pregnancies [De Stefano, et al., Thromb Haemost74:793–794, 1995].

Thus, the risk of venous thromboembolic complications occurring inpregnant women is a major concern, especially in women who have ahistory of thromboembolic events. Although the possibility of severecomplications such as preeclampsia or DIC is relatively low, it has beensuggested that it is essential to start therapy of DIC as soon as it hasbeen diagnosed by onset of inhibition of the activated coagulationsystem [Rathgeber, et al., Anasth Intensivther Notfallmed 25:206–211,1990]. The complications of preeclampsia or DIC is analogous to thesituation that occurs in sepsis in that there is a hypercoagulable stateand a decrease in the levels of protein C (Levi, et al., N. Engl. J.Med. 341:586–592, 1999).

Major Surgery/Trauma

Patients recovering from major surgery or accident trauma frequentlyencounter blood coagulation complications as a result of an inducedhypercoagulable state [Watkins, et al., Klin Wochenschr 63:1019–1027,1985]. Hypercoagulable states are increasingly recognized as causes ofvenous thromboembolism in surgical patients [Thomas, et al., Am J Surg.158:491–494, 1989; LeClerc, J. R., Clin Appl Thrombosis/Hemostasis3(3):153–156, 1997]. Furthermore, this hypercoagulable state can lead tocomplications with DIC-like symptoms, which is infrequently encounteredbut, nonetheless, is devastating and often fatal when it occurs.[Collins, et al., Am J Surg. 124:375–380, 1977].

In addition, patients undergoing coronary artery bypass grafting (CABG)[Menges, et al., J Cardiothor Vasc An. 10:482–489, 1996], major spinalsurgery [Mayer, et al., Clin Orthop. 245:83–89, 1989], major abdominalsurgery [Blamey, et al., Thromb Haemost. 54:622–625, 1985], majororthopedic surgery or arthroplastic surgery of the lower extremities[LeClerc, 1997], or other types of surgery [Thomas, et al., Am J Surg.158:491–494, 1989], occasionally develop venous thromboemboliccomplications. Additionally, investigators in Japan have proposedtreating microvascular thrombosis associated with spinal cord injury[patent application JP8325161A] with plasma derived PCZ at a dose of1–10 mg/day for an adult, or preferably, 2–6 mg divided by 1–2 times tobe administered as a bolus or by intravenous infusion.

It has been suggested that anti-coagulant therapy is important as aprophylactic therapy to prevent venous thromboembolic events in majorsurgery or trauma patients [Thomas, et al., 1989; LeClerc, 1997]. Forexample, many patients who succumb from pulmonary embolism have noclinical evidence of preceding thromboembolic events and die before thediagnosis is made and the treatment is instituted [LeClerc, 1997].Existing prophylactic methods e.g., warfarin, low molecular weightheparins, have limitations such as residual proximal thrombosis or theneed for frequent dose adjustments.

Ards

Adult respiratory distress syndrome [ARDS] is characterized by lungedema, microthrombi, inflammatory cell infiltration, and late fibrosis.Pivotal to these multiple cellular and inflammatory responses is theactivation of coagulation resulting in a hypercoagulable state. CommonARDS-associated coagulation disorders include intravascular coagulationand inhibition of fibrinolysis. Fibrin formed by the activation of thecoagulation system and inhibition of fibrinolysis presumably contributesto the pathogenesis of acute lung injury. Sepsis, trauma and othercritical diseases are important risk factors that lead to ARDS[Hasegawa, et al., Chest 105(1):268–277, 1994].

ARDS is associated with an activation of coagulation and inhibition offibrinolysis. Considerable clinical evidence exists for the presence ofpulmonary vascular microemboli which is analogous to thehypercoagulation that is present in DIC. Therefore, a need currentlyexists for an effective treatment of this hypercoagulable stateassociated with ARDS.

The present invention provides a method of treating human patients witha hypercoagulable state or protein C deficiency which comprisesadministering to said patient aPC and PCZ.

The invention further provides a method of treating human patients witha hypercoagulable state or protein C deficiency which comprisesadministering to said patient an effective amount of aPC and PCZ toachieve activated Protein C plasma levels in the range of 2 ng/ml to 200ng/ml.

Another aspect of this invention provides methods for treating vascularocclusive disorders and acquired and congenital hypercoagulable states,including sepsis (including, severe sepsis and septic shock),disseminated intravascular coagulation, purpura fulminans, major trauma,major surgery, burns, adult respiratory distress syndrome (ARDS),melioidosis, preeclampsia, eclampsia, amniotic fluid embolism, placentaabruption, transplantations, deep vein thrombosis, heparin-inducedthrombocytopenia, sickle cell disease, thalassemia, viral hemorrhagicfever, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome,acute coronary syndromes (ACS; e.g., unstable angina, myocardialinfarction) and acquired and congenital protein C deficiency,characterized by administering a combination of aPC and PCZ.

Inventive formulations are also provided. Preferably, said formulationsare lyophilized, adapted for parenteral administration and comprise aPCand PCZ as the active ingredients in any ratio, together with apharmaceutical carrier. For example, preferred ratios of PCZ:aPCinclude: 5% by weight PCZ: 95% by weight aPC; 10% by weight PCZ: 90% byweight aPC; 15% by weight PCZ: 85% by weight aPC; 20% by weight PCZ: 80%by weight aPC; 25% by weight PCZ: 75% by weight aPC; 30% by weight PCZ:70% by weight aPC; 35% by weight PCZ: 65% by weight aPC; 40% by weightPCZ: 60% by weight aPC; 45% by weight PCZ: 55% by weight aPC; and 50% byweight PCZ: 50% by weight aPC; 60% by weight PCZ: 40% by weight aPC; 70%by weight PCZ: 30% by weight aPC; 80% by weight PCZ: 20% by weight aPC;90% by weight PCZ: 10% by weight aPC; 95% by weight PCZ: 5% by weightaPC; 99% by weight PCZ: 1% by weight aPC. Preferably, said formulationsfurther comprise a salt (e.g., sodium chloride, calcium chloride,potassium chloride), a bulking agent (e.g., mannitol, trehalose,raffinose, sucrose, or mixtures of various bulking agents), a buffer(e.g., Tris-acetate, sodium citrate, and sodium phosphate, or mixturesof buffers), and optionally, a stabilizer (e.g., albumin). Theformulations herein, upon reconstitution with an acceptable diluent,have preferred pH ranges and values, as follows: pH of about 6.0 toabout 8.0; pH of about 6.0 to about 7.0; pH of about 6.3 to about 6.7.The inventive formulations are preferably adapted for use in treatingthe methods described herein. The formulations may be prepared viamultiple methods, including by lyophilizing a solution comprising aPC,PCZ, and a bulking agent. Preferably, the solution to be lyophilizedfurther comprises a salt and/or a buffer, and optionally a stabilizingagent. Also included, is an article of manufacture, comprising packagingmaterial and activated Protein C contained within said packagingmaterial, wherein the packaging material comprises a label whichindicates that activated Protein C can be used in combination withprotein C zymogen for treating a disease or condition selected from:sepsis, severe sepsis, septic shock, disseminated intravascularcoagulation, purpura fulminans, major trauma, undergoing or recoveringfrom surgery, burns, adult respiratory distress syndrome, bone marrowand other organ transplantations, deep vein thrombosis, heparin-inducedthrombocytopenia, sickle cell disease, thalassemia, viral hemorrhagicfever, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome,unstable angina, myocardial infarction, meningococcemia, melioidosis,complications during pregnancy, preeclampsia, eclampsia, amniotic fluidembolism, placental abruption, and chemotherapy.

For purposes of the present invention, as disclosed and claimed herein,the following terms are as defined below.

APC, aPC, or activated Protein C refer to the activated human protein Cmolecule, whether plasma derived or produced by recombinant ortransgenic means. Recombinant and transgenic activated Protein C may beproduced by activating the human protein C zymogen in vitro or by directsecretion or production of the activated form of protein C. Protein Cmay be produced in cells, eukaryotic cells, transgenic animals, ortransgenic plants, including, for example, secretion from human kidney293 cells as a zymogen then purified and activated by techniques knownto the skilled artisan.

Treating—describes the management and care of a patient for the purposeof combating a disease, condition, or disorder. Treating may alsoinclude prophylaxis, preventing or prophylactic administration toprevent the onset of the symptoms or complications of the disease,condition, or disorder.

Continuous infusion—continuing substantially uninterrupted theintroduction of a solution into a vein for a specified period of time.

Bolus injection—the injection of a drug in a defined quantity (called abolus) over a period of time, for example, up to about 1–120 minutes.

Suitable for administration—a formulation or solution that isappropriate to be given as a therapeutic agent.

Receptacle—a container such as a vial or bottle that is used to receivethe designated material, i.e., PCZ or aPC or combinations thereof.

Unit dosage form—refers to physically discrete units suitable as unitarydosages for human subjects, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect, in association with a suitable pharmaceuticalexcipient.

Hypercoagulable states—excessive coagulability associated withdisseminated intravascular coagulation, pre-thrombotic conditions,activation of coagulation, or congenital or acquired deficiency ofclotting factors such as PCZ.

Zymogen—Protein C zymogen, as used herein, refers to secreted, inactiveforms, whether one chain or two chains, of protein C.

Juvenile—a human patient including but not restricted to newborns,infants, and children younger than 18 years of age.

Effective amount—a therapeutically efficacious amount of apharmaceutical compound or compounds, particularly aPC and/or PCZ.

Purpura fulminans—ecchymotic skin lesions, fever, hypotension associatedwith bacterial sepsis, viral, bacterial or protozoan infections.Disseminated intravascular coagulation is usually present.

Sepsis-refers to a systemic response to infection or suspected infectioncomplicated by one or more organ failures. For purposes of thisapplication, the term “sepsis” also includes severe sepsis (sepsis withevidence of one of more organ failure. Organs can be cardiovascular,metabolic, mental status/central nervous system,hematologic/coagulation, renal, respiratory, hepatic) and septic shock(defined as hypotension or hypoperfusion to end organs).

Protein C deficiency can be determined and defined by two primary meansdepending upon the availability of patient data. For a patient whosenormal plasma protein C level is known, or if serial plasma protein Clevel is performed on a patient (for example, every 6–12 hours),acquired protein C deficiency can be defined as a 10% or greaterdecrease from either the patient's own known normal level or from arecent protein C level value that was within a normal range. When apatient's normal plasma protein C level is not known or cannot beobtained (for example, a patient upon admission to the hospital isalready presenting with clinical symptoms of severe sepsis or laboratorytests indicate a hypercoagulable state), then the acquired protein Cdeficiency is generally defined as below the lower limit of the normalrange of protein C as established or used by the laboratory thatperforms the protein C assay.

The present invention relates to the treatment or prevention ofhypercoagulable states or acquired protein C deficiency, particularlywhen such states or deficiency is associated with: sepsis,transplantation, burns, pregnancy, major surgery, trauma, or ARDS, withaPC and PCZ. The aPC and PCZ can be made by techniques well known in theart utilizing eukaryotic cell lines, transgenic animals, or transgenicplants. Skilled artisans will readily understand that appropriate hosteukaryotic cell lines include but are not limited to HEPG-2, LLC-MK₂,CHO-K1, 293, or AV12 cells, examples of which are described by Grinnellin U.S. Pat. No. 5,681,932, herein incorporated by reference.Furthermore, examples of transgenic production of recombinant proteinsare described by Drohan, et al., in U.S. Patent No. 5,589,604 andArchibald, et al., U.S. Patent No. 5,650,503, herein incorporated byreference. U.S. Patent No. 5,009,889 (incorporated by reference herein)describes various procedures for isolating protein C from plasma. Foradditional examples of methods to prepare PCZ and aPC, and formulationscontaining the same, See: U.S. Pat. 5,580,962 (columns 3 and 4); U.S.Pat. 5,831,025; European Patent Application 0662513A1; U.S. Pat. No.5,093,117; and U.S. Pat. No. 5,084,273.

To be fully active, the aPC made by any of these methods must undergopost-translational modifications such as the carboxylation of theside-chain of nine glutamate residues to gamma-carboxy-glutamates(gamma-carboxylation, i.e., Gla content), the hydroxylation of the sidechain of one aspartate residue to erythro-beta-hydroxy-Asp(beta-hydroxylation), the glycosylation of the side chain of fourasparagine residues to Asn-linked oligosaccharides (glycosylation), theremoval of the leader sequence (42 amino acid residues) and removal ofthe dipeptide Lys 156-Arg 157. Without such post-translationalmodifications, aPC is not fully functional or is non-functional.

The following Methods, Preparations and Examples are illustrative andare not intended to limit the invention in any way.

Method for Determining Protein C Levels in Patient Plasma Samples

Protein C levels can be determined in patient citrated plasma samplesusing appropriately approved diagnostic kits by appropriately certifiedlaboratories and trained laboratory technicians. There are generallythree types of diagnostic kits for measuring protein C levels fromvarious commercial companies. One is to measure the antigenic level ofprotein C in plasma by an ELISA type methodology. The other two methodsare to measure the protein C activity level. Protein C is firstconverted to activated Protein C, generally using a protease extractedfrom snake venom, and then the activity is measured either by itsamidolytic activity (amidolytic activity kit) or by its anticoagulantactivity (clotting activity kit). Any of the three diagnostic kits canbe used to determine protein C deficiency in patients. For acquiredprotein C deficiency or an acquired hypercoagulable state where liverdysfunction may be involved, the preferred or more clinically relevantmethod for determining the protein C level in a patient is the clottingactivity diagnostic kit.

Protein C levels are usually measured in patient citrated plasma. Apatient's blood sample is usually collected into either a 2.8 ml(pediatric size) or 4.5 ml vacutainer containing either 3.2% or 3.8%citrate. The blood sample can be obtained either via veni-puncture orvia a central line. If heparin contamination cannot be avoided whencollecting the blood sample, for example, via central line, then onlythe antigenic method can be used to measure accurately the protein Clevels in that sample. The citrated blood sample is centrifuged at about2000×g for 10 to 20 minutes. The citrated plasma, which is thesupernatant can be removed and used for the protein C level measurement.

The measurement of plasma protein C levels using any one of the threekinds of diagnostic kits can be carried out using manual, semi-automatedor automated equipment. Appropriately certified laboratories andtechnicians usually have detailed standard operating procedures forperforming the protein C assays. The standard operating proceduresshould include appropriate validation of the assays and the equipmentused prior to assaying patient samples. In general, human plasmastandard samples with known levels of protein C are used to calibrateand validate the assay and equipment. The intra- and inter-day variationof the assay results using these known standards should be less than 10%CV.

Determination of Normal (100%) Level of Human Plasma Protein C

Normal (100%) of human plasma protein C level is defined as the amountof protein C in a pooled normal plasma sample. This pooled normal humanplasma sample can be the established WHO international standard (1 ml ofpooled citrated plasma). This can also be supplied as part of thecommercially available protein C diagnostic kit. This is usuallyprepared by combining citrated plasma from 20 to more than a hundrednormal human donors. The pooled plasma is then aliquoted and generallystored as a 1 ml lyophilized or frozen liquid in vials with specifiedexpiration date.

Determination of Normal Range of Human Plasma Protein C

The normal range of human plasma protein C is generally determined byeach laboratory as part of the validation for determining human plasmaprotein C level for the purpose of providing clinical diagnosis of thepatient by the clinical staff. The normal range will vary slightly fromlaboratory to laboratory depending upon the diagnostic kit/method andthe equipment used to perform the protein C assay. A concentrationstandard curve is determined with the standards provided and theprocedure accompanied by the diagnostic kit and the equipment. Thenormal range is determined by measuring the concentration of protein Cin a citrated plasma sample from about 30–120 normal healthy individualdonors who are not on any medications that can affect their bloodclotting chemistry. The lower and upper limit of the normal range aredetermined by taking two standard deviations from the mean (if the rangeis of normal distribution) or the median (if the range is not of normaldistribution). The lower limit of normal range for adult human (≧18years of age) is usually around 60–80% of pooled normal plasma. Theupper limit of normal range for adult human (≧18 years of age) isusually around 140–180%. A normal new born usually has a plasma proteinC level of about 30–40% of an normal adult. By about 1 year of age, theplasma protein C level in a normal child will reach to about the lowerlimit of a normal adult. Thus the normal range in children is differentfrom that of adult and needs to be determined separately.

Preparation 1 Preparation of Human PCZ

Recombinant human PCZ was produced in Human Kidney 293 cells bytechniques well known to the skilled artisan such as those set forth inYan, U.S. Pat. No. 4,981,952, the entire teaching of which is hereinincorporated by reference. The gene encoding human protein C isdisclosed and claimed in Bang, et al., U.S. Pat. No. 4,775,624, theentire teaching of which is incorporated herein by reference. Theplasmid used to express human protein C in 293 cells was plasmid PLPCwhich is disclosed in Bang, et al., U.S. Pat. No. 4,992,373, the entireteaching of which is incorporated herein by reference. The constructionof plasmid pLPC is also described in European Patent Publication No. 0445 939, and in Grinnell, et al., 1987, Bio/Technology 5:1189–1192, theteachings of which are also incorporated herein by reference. Briefly,the plasmid was transfected into 293 cells, then stable transformantswere identified, subcultured and grown in serum-free media. Afterfermentation, cell-free medium was obtained by microfiltration.

The human protein C was separated from the culture fluid by anadaptation of the techniques of Yan, U.S. Pat. No. 4,981,952, the entireteaching of which is herein incorporated by reference. The clarifiedmedium was made 4 mM in EDTA before it was absorbed to an anion exchangeresin (Fast-Flow Q, Pharmacia). After washing with 4 column volumes of20 mM Tris, 200 mM NaCl, pH 7.4 and 2 column volumes of 20 mM Tris, 150mM NaCl, pH 7.4, the bound recombinant human PCZ was eluted with 20 mMTris, 150 mM NaCl, 10 mM CaCl₂, pH 7.4. The eluted protein was greaterthan 95% pure after elution as judged by SDS-polyacrylamide gelelectrophoresis.

Further purification of the protein was accomplished by making theprotein 3 M in NaCl followed by adsorption to a hydrophobic interactionresin (Toyopearl Phenyl 650 M, TosoHaas) equilibrated in 20 mM Tris, 3 MNaCl, 10 mM CaCl₂, pH 7.4. After washing with 2 column volumes ofequilibration buffer without CaCl₂, the recombinant human protein C waseluted with 20 mM Tris, pH 7.4.

The eluted protein was prepared for activation by removal of residualcalcium. The recombinant human protein C was passed over a metalaffinity column (Chelex-100, Bio-Rad) to remove calcium and again boundto an anion exchanger (Fast Flow Q, Pharmacia). Both of these columnswere arranged in series and equilibrated in 20 mM Tris, 150 mM NaCl, 5mM EDTA, pH 6.5. Following loading of the protein, the Chelex-100 columnwas washed with one column volume of the same buffer beforedisconnecting it from the series. The anion exchange column was washedwith 3 column volumes of equilibration buffer before eluting the proteinwith 400 mM NaCl, 20 mM Tris-acetate, pH 6.5. Protein concentrations ofrecombinant human PCZ and recombinant aPC solutions were measured by UV280 nm extinction E^(0.1%=)1.85 or 1.95, respectively.

Preparation 2 Activation of Recombinant Human PCZ

Bovine thrombin was coupled to Activated CH-Sepharose 4B (Pharmacia) inthe presence of 50 mM HEPES, pH 7.5 at 4° C. The coupling reaction wasdone on resin already packed into a column using approximately 5000units thrombin/ml resin. The thrombin solution was circulated throughthe column for approximately 3 hours before adding MEA to aconcentration of 0.6 ml/l of circulating solution. The MEA-containingsolution was circulated for an additional 10–12 hours to assure completeblockage of the unreacted amines on the resin. Following blocking, thethrombin-coupled resin was washed with 10 column volumes of 1 M NaCl, 20mM Tris, pH 6.5 to remove all non-specifically bound protein, and wasused in activation reactions after equilibrating in activation buffer.

Purified PCZ was made 5 mM in EDTA (to chelate any residual calcium) anddiluted to a concentration of 2 mg/ml with 20 mM Tris, pH 7.4 or 20 mMTris-acetate, pH 6.5. This material was passed through a thrombin columnequilibrated at 37° C. with 50 mM NaCl and either 20 mM Tris pH 7.4 or20 mM Tris-acetate pH 6.5. The flow rate was adjusted to allow forapproximately 20 min. of contact time between the PCZ and thrombinresin. The effluent was collected and immediately assayed for amidolyticactivity. If the material did not have a specific activity (amidolytic)comparable to an established standard of aPC, it was recycled over thethrombin column to activate the PCZ to completion. This was followed by1:1 dilution of the material with 20 mM buffer as above, with a pH ofanywhere between 7.4 or 6.0 (lower pH being preferable to preventautodegradation) to keep the aPC at lower concentrations while itawaited the next processing step.

Removal of leached thrombin from the aPC material was accomplished bybinding the aPC to an anion exchange resin (Fast Flow Q, Pharmacia)equilibrated in activation buffer (either 20 mM Tris, pH 7.4 orpreferably 20 mM Tris-acetate, pH 6.5) with 150 mm NaCl. Thrombin passesthrough the column and elutes during a 2–6 column volume wash with 20 mMequilibration buffer. Bound aPC is eluted with a step gradient using 400mM NaCl in either 5 mM Tris-acetate, pH 6.5 or 20 mM Tris, pH 7.4.Higher volume washes of the column facilitated more complete removal ofthe dodecapeptide. The material eluted from this column was storedeither in a frozen solution (−20° C.) or as a lyophilized powder.

The amidolytic activity (AU) of aPC was determined by release ofp-nitroanaline from the synthetic substrateH-D-Phe-Pip-Arg-p-nitroanilide (S-2238) purchased from Kabi Vitrum usinga Beckman DU-7400 diode array spectrophotometer. One unit of aPC wasdefined as the amount of enzyme required for the release of 1 μmol ofp-nitroaniline in 1 min. at 25° C., pH 7.4, using an extinctioncoefficient for p-nitroaniline at 405 nm of 9620 M⁻¹ cm⁻¹.

The anticoagulant activity of aPC was determined by measuring theprolongation of the clotting time in the activated partialthromboplastin time (APTT) clotting assay. A standard curve was preparedin dilution buffer (1 mg/ml radioimmunoassay grade BSA, 20 mM Tris, pH7.4, 150 mM NaCl, 0.02% NaN₃) ranging in protein C concentration from125–1000 ng/ml, while samples were prepared at several dilutions in thisconcentration range. To each sample cuvette, 50 μl of cold horse plasmaand 50 μl of reconstituted activated partial thromboplastin time reagent(APTT Reagent, Sigma) were added and incubated at 37° C. for 5 min.After incubation, 50 μl of the appropriate samples or standards wereadded to each cuvette. Dilution buffer was used in place of sample orstandard to determine basal clotting time. The timer of the fibrometer(CoA Screener Hemostasis Analyzer, American Labor) was started upon theaddition of 50 μl, 37° C., and 30 mM CaCl₂ to each sample or standard.aPC concentration in samples is calculated from the linear regressionequation of the standard curve. Clotting times reported here are theaverage of a minimum of three replicates, including standard curvesamples.

The above descriptions enable one with appropriate skill in the art toprepare PCZ and aPC for use in treating the hypercoagulable states oracquired protein C deficiency as described herein.

EXAMPLE 1 Human Plasma Levels of aPC

Six human patients received an intravenous infusion of aPC at 1 mg/m²/hror about 0.024 mg/kg/hr over a 24 hour period. The aPC administered wasa lyophilized formulation containing 10 mg aPC, 5 mM Tris acetate bufferand 100 mM sodium chloride reconstituted with two ml of water andadjusted to pH 6.5.

Plasma concentrations of aPC were measured using anImmunocapture-Amidolytic Assay. Blood was collected in the presence ofcitrate anticoagulant and benzamidine, a reversible inhibitor of aPC.The enzyme was captured from plasma by an aPC specific murine monoclonalantibody, C3, immobilized on a microtiter plate. The inhibitor wasremoved by washing and the amidolytic activity of aPC was measured usingan oligopeptide chromogenic substrate. Following incubation for 16–20hours at 37° C., the absorbance was measured at 405 nm and data areanalyzed by a weighted linear curve-fitting algorithm. aPCconcentrations were estimated from a standard curve ranging inconcentrations from 0–100 ng/ml. The limit of quantitation of the assaywas 1.0 ng/ml. The aPC dose levels and plasma concentrations weremeasured at about 24 hours. The dose of 0.024 mg/kg/hr yields a plasmaconcentration of about 50 ng/ml at 24 hours.

EXAMPLE 2 Double-blinded Placebo-controlled Trial in Human Patients WithSepsis, Stage 1

This protocol is a two-stage, double-blinded placebo-controlled trial inpatients with severe sepsis. In Stage 1, a total of 72 patients wereinfused for 48 hours with recombinant human aPC.

Entry criteria included three of the four commonly accepted criteria forsepsis (heart rate, respiratory effort, increased/decreased temperature,increase/decrease white blood cell count). The patients also had todemonstrate some degree of organ dysfunction defined as either shock,decreased urine output, or hypoxemia. Four different doses wereutilized; 12, 18, 24, 30 μg/kg/hr. The aPC was infused for 48 hours by acontinuous infusion method. The primary endpoints of this study were:safety as a function of dose and dose duration, and the ability of aPCto correct coagulopathy as a function of dose and dose duration.

Mortality information includes all doses, even the lowest doses, unlessotherwise specified. It is important to note that the placebo mortalityobserved in this study is consistent with anticipated placebo mortality.A 28 day all cause mortality was the end-point in patients receivingplacebo vs. patients receiving aPC.

The overall observed placebo mortality rate was 38% (10/26) and theoverall observed aPC mortality rate was 20% (9/46). A subgroup involvingonly the top two doses of aPC (24 and 30 μg/kg/hr) vs. placebo patientshad an observed mortality rate of 13% (3/24).

A second subgroup analysis included patients with an acquired protein Cdeficiency, defined as a baseline protein C activity of less than 60%.Of the 64 patients that have baseline protein C activity data available,61 patients or 95%, had an acquired protein C deficiency at the time ofentry into the study. The observed placebo mortality rate for protein Cdeficient patients was 41% (9/22) and the observed aPC mortality ratefor protein C deficient patients was 18% (7/39).

A significant piece of information suggesting that treatment with aPC isof benefit with patients with severe sepsis includes the mean time todeath in placebo patients vs. treated patients. Of the 10 patients whodied in the placebo group, the mean time to death was 6 days. In the aPCtreated patients, the mean time to death was 14 days. Additionally, 4 ofthe 9 patients who died in the aPC treatment arm survived 21 or moredays and subsequently succumbed to an event unrelated to their firstepisode of sepsis. Two of the four late deaths occurred in the low dosegroup (12 μg/kg/hr). Both of these patients remained in the ICU andmechanically ventilated the entire duration of the study until theirdeath (day 27). The other two patients with late deaths were in thehigher dose group (30 μg/kg/hr). Both of these patients showed initialimprovement. Within two weeks both were off mechanical ventilation andtransferred from the ICU. One patient died a week later from sepsisinduced respiratory distress after requesting a “do not resuscitate”(DNR) order enacted. The second patient died on day 28 after sufferingan episode of pulmonary insufficiency related to a second episode ofsepsis. This patient had also requested DNR status and therefore was notreintubated. It should be noted that retreatment with aPC of patientsthat develop a second episode of severe sepsis during the 28 day studywas not approved under the treatment protocol.

The mortality information in this study is surprising and unexpected. Noother double-blinded, placebo controlled sepsis study has generated datademonstrating such a marked reduction in 28 day all cause mortality.

The administration of PCZ and aPC in order to practice the presentmethods of therapy is carried out by administering an effective amountof each chosen compound (PCZ and/or aPC) to the patient in need thereof.The effective amount of each individual compound, and the appropriatedosing regimen, is determined, in the final analysis, by the physicianin charge of the case, but depends on factors such as the exact diseaseor diseases to be treated, the severity of the disease and otherdiseases or conditions from which the patient suffers, the specificroute of administration, other drugs and treatments which the patientmay concomitantly require, and no doubt other factors in the physician'sjudgement.

Preferably the aPC is administered by continuous infusion for about 24to about 144 hours at a dosage of about 1 μg/kg/hr to about 50 μg/kg/hr.More preferably, the amount of aPC administered will be about 4 μg/kg/hrto about 48 μg/kg/hr. Even more preferably the amount of aPCadministered will be: about 6 μg/kg/hr to about 44 μg/kg/hr; about 8μg/kg/hr to about 40 μg/kg/hr; about 10 μg/kg/hr to about 36 μg/kg/hr;about 12 μg/kg/hr to about 34 μg/kg/hr; about 14 μg/kg/hr to about 30μg/kg/hr; about 16 μg/kg/hr to about 24 μg/kg/hr; about 18 μg/kg/hr toabout 20 μg/kg/hr; about 6 μg/kg/hr to about 22 μg/kg/hr; or about 10μg/kg/hr to about 20 μg/kg/hr; or about 5 μg/kg/hr to about 25 μg/kg/hr;or about 5 μg/kg/hr to about 30 μg/kg/hr. The preferred amounts of PCZadministered will be about 4 μg/kg/hr to about 800 μg/kg/hr; about 20μg/kg/hr to about 600 μg/kg/hr; 40 μg/kg/hr to about 400 μg/kg/hr; 60μg/kg/hr to about 120 μg/kg/hr; 80 μg/kg/hr to about 200 μg/kg/hr; or100 μg/kg/hr to about 150 μg/kg/hr; or about 25 μg g/kg/hr to 100μg/kg/hr (infusion only), more preferably about 40 μg/kg/hr to about 80μg/kg/hr (infusion); and most preferably about 90 μg/kg/hr, infusiononly. Alternatively, a bolus may be administered at various intervalsbefore during or after discontinuation of the infusion. The bolus ispreferably in the range of about or about 25 to 100 μg/kg/hr to about500 μg/kg/hr (bolus followed by infusion); or about 200 μg/kg to 2000μg/kg once daily.

A physician may dose the PCZ and aPC to achieve preferred PCZ and/or aPCplasma levels. Should the physician desire rapid aPC plasma levels, aPCwill be administered in a bolus or in an increased amount. aPC will alsobe preferred when it appears that a patient is not responding to PCZ,possibly because of a perceived or documented inability of a patient toconvert PCZ to aPC. Examples of preferred protein C plasma level-rangesinclude: about 10 ng/ml to about 180 ng/ml; about 25 ng/ml to about 160ng/ml; about 25 ng/ml to about 100 ng/ml; about 30 ng/ml to about 140ng/ml; about 40 ng/ml to about 120 ng/ml; about 40 ng/ml to about 100ng/ml; and about 40 to about 80 ng/ml. Again, although the preferreddoses and plasma ranges are stated herein, various boluses of PCZ and/oraPC may be used at various intervals, as is preferred in the judgementof the physician.

For examples of dosing regimens of aPC and PCZ noted in literature andpatent documents, Table I sets forth normalized dose levels of severalstudies in humans or non-human primates. The human studies were doneutilizing plasma derived PCZ while the non-human primate study utilizedrecombinant human aPC.

TABLE I REFERENCE PUBLISHED DOSE NORMALIZED DOSE⁺ Taylor, et al., IVadministration of between 2 and 64 μg aPC/kg/minute; 120 μ/kg/hr to U.S.Pat. No. a bolus of between 1 and 10 mg aPC may be given 3800 ug/kg/hr5,009,889 additionally. infused for 8 to 10 [column 5, lines 14–19]hours Rivard, et al., IV administration at a dose of 100 IU*/kg plasma400 ug/kg in 15 to J. Ped. 126: 646, derived protein C zymogen during a15 to 20 minute 20 minutes 1995 period every 6 hours during the acutephase and then 1 to 2 times a day for 9 days. [p.648, column 1, 1^(st)paragraph] Gerson, et al., IV administration at a bolus dose of 70IU*/kg plasma 280 ug/kg bolus Ped. 91: 418–422, derived protein Czymogen every 6 hours. Subsequently, every 6 hours, then 1993 continuousinfusion of 10 IU/kg/hr for 11 days was continuous infusion given. of 40ug/kg/hr for [p.419, column 2, 1^(st) paragraph] 11 days Rintala, etal., IV administration was started 3 hours after admission 400 ug/kgbolus every Lancet 347: 1767, and continued for 7 days. 100 IU*/kgplasma derived 6 to 8 hours for 7 1996 protein C zymogen every 6 hoursand later adjusting days dose to plasma protein C activity. [p.1767,column 2, 2^(nd) paragraph] Ettingshausen, et Plasma-derived humanprotein C treatment was initiated 320 to 480 μg/kg al., Semin. on theday of admission and continued for 1 to 16 days. bolus + 50 μg/kg/hrThromb. Hemost. Each patient received an initial bolus of 80 to 120infusion for 3 days 25: 537–541, 1999 IU*/kg or protein C followed by aone hour infusion of (median) with a 50 IU*/kg protein C, given onceevery 4 hours during range of 1 to 16 the early acute phase of theillness. The frequency of days. the hourly infusion was adjusted withonce or twice daily monitoring of the endogenous protein C level to aimat maintaining an endogenous protein C level in the normal range.[p.538, column 2, 2^(nd) paragraph] Betrosian, et al. Plasma-derivedhuman protein C treatment was initiated 67 μg/kg/hr was Crit. Care Med.48 hours after admission and continued for 3 days. The given the first24 27: 2849–2850, patient was given 100 IU*/kg by IV administration oncehours and 40 1999 every 6 hours the first day and at 60 IU*/kg every 6μg/kg/hr was given hours the subsequent 2 days. The endogenous protein Cthe 2 additional level was maintained at 85 to 140% during the therapy.days. [p.2850, column 1, 1^(st) paragraph] Veldman et al.,Plasma-derived human protein C treatment was initiated 10 μg/kg/hr for18 Bone Marrow the same time as heparin and t–PA treatment for 18 daysTransplant. days. Protein C was given as intravenous infusion of 21:S238, 1998 60 IU*/kg/24 hours. [p.S238, column 2, abstract # 834]Toupance et al., Plasma-derived human protein C was given 4 to 8μg/kg/hr for Transpl. Int. prophylactically before and after renaltransplant at 10 to 17 days in a 7: 144–145, 1994 50 IU*/kg twice dailyfor 10 days and once daily for 7 prophylactic and more days. During thenext 6 months after renal treatment modes transplant, protein C wasgiven as treatment for several thrombotic crisis at the same dose forabout 10 days each time. [p.145, column 1, 2^(nd) paragraph) Favier, etal., Plasma-derived human protein C treatment was initiated 4 μg/kg/hrfor 6 Hematol. Cell 6 days after admission and was given intravenouslyat days, stopped for 4 Therapy 40: 67–70, 10 IU*/kg/10 hours for 6 days.The treatment was days and treated 1998 stopped for 4 days and resumedfor 5 days. again at the same [p.69, column 1, 1^(st) and 2^(nd)paragraphs] dose for 5 days Minford, et al., Plasma-derived humanprotein C treatment was given by 1000 μg/kg Br. J. Haematol.subcutaneous infusion over 2 hours via a Graseby subcutaneous every 93:215–216, 1996 syringe pump at 250 IU*/kg every 48 hours for long term 48hours for long therapy. This raised the protein C level in the termtherapy patient at a peak of about 90% to a nadir of about 25% inbetween dosing. [p.215, column 2, 1^(st) paragraph] San–Rodriguez, etPlasma-derived human protein C treatment was given by 1400 μg/kg al.,Br. J. subcutaneous route at 350 IU*/kg every 48 hours for 9subcutaneous every Haematol. 102: 16, months and beyond for long termtherapy. 48 hours for long 1998 [p.16, column 2, #0–0059] term therapySmith, et al., Plasma-derived human protein C treatment was initiated400 ug/kg bolus + 60 Lancet, 350: 1590– 8 to 72 hours after admissionand continued for 1 to 8 ug/kg/hr for 5.7 1593, 1997 days. Each patienthad a test dose of 40 IU*/kg over days (mean) with a 10 min. Then aloading dose of 100 IU*/kg plasma range of 1 to 8 days derived protein Czymogen followed by a continuous infusion of 15* IU/kg. [p.1591, column2, 4^(th) paragraph] Fujiwara, et al., The usual dose is 20–1000 U**plasma derived 4 ug/kg to Japanese Patent APC/kg body weight/day, ormore preferably 50–300 200 ug/kg. JP7097335A U/kg with dividedadministration of 1–2 times. An infusion time was As the method ofadministration, it is most not given. appropriate to use intravenousinfusion. [p.9, paragraph 0016] Okajima, et al., The effective dose ofplasma derived PC or APC is 42 ug/hr to Japanese Patent 1–10 mg/day foran adult, or preferably 2–6 mg to 420 ug/hr JP 8325161A be administereddivided 1–2 times. As the method of administration, one can use bolusadministration (in a single administration) or intravenous infusion.[p.10, paragraph 0013] Okajima, et al., Administration of plasma derivedaPC (3 †mg/day 2 ug/kg/hr and Amer. J of for 2 days, followed by 6mg/day for 3 days). 4 ug/kg/hr. Hematology, [p.278, column 1, 1^(st)full paragraph] 33: 277–278 (1990) Kobayashi, et al., Plasma-derivedhuman aPC was given at 5000 to 21 to 42 μg/hr for 2 Thromb. Haemost.10000 units** over 2 days. days 82: 1363, 1999 [p.1363, column 1, 2^(nd)paragraph] Wada, et al., Am. Plasma-derived human APC was given at 400033 μg/hr for 6 days J. Hematol. units**/day for 6 days. 44: 218–219,1993 [p.219, column 1, 1^(st) paragraph] Wada, et al., Plasma-derivedhuman APC was given at 100 to 300 0.8 to 2.5 μg/kg/hr Blood 94: 28a,1999 units**/kg for 3 to 6 days. This dose is not for 3 to 6 dayssufficient for treating purpura fulminans. [p.28a, column 2, #111] Bang,et al., U.S. The dose of activated Protein C ranges from 1–10 1.8 to 18ug/kg/hr Pat. No. 4,775,624 mg as a loading dose followed by acontinuous An infusion time was infusion in amounts ranging from 3–30mg/day. not given. [column 19, lines 55–59] *the normalized dose is aconversion of the reported dose to the equivalent ug/kg/hr designation.•1 IU is equivalent to approximately 4 ug of PC **1 U is defined as theamount which doubles the activated prothrombin time (APTT) in normalhuman plasma. This converts to approximately 5 Units/ug APC.

Another embodiment of the present invention is a pharmaceuticalformulation comprising an effective amount of aPC and PCZ. The aPC andPCZ can be formulated together in a formulation according to knownmethods to prepare pharmaceutically useful compositions. Preferably theaPC and PCZ formulation will comprise, consist essentially of, orconsist of, a salt; such as, sodium, or calcium or potassium chloride, abulking agent, preferably sucrose, mannitol, dextran, trehalose, orraffinose; and a buffer, preferably a phosphate or sodium chloridebuffer, and most preferably a buffer selected from Tris-acetate, sodiumcitrate, sodium phosphate and combinations thereof. A stabilizer, suchas albumin, may also be added. To prevent or reduce the autodegration ofPCZ and aPC, purification and/or formulation may be performed in thepresence of a denaturing agent. For example, urea may be used atconcentrations of about 3 M. The formulation is preferably a lyophilizedformulation. The active ingredients may be in any ratio, for example,preferred ratios of PCZ: aPC include: 5% by weight PCZ: 95% by weightaPC; 10% by weight PCZ: 90% by weight aPC; 15% by weight PCZ: 85% byweight aPC; 20% by weight PCZ: 80% by weight aPC; 25% by weight PCZ: 75%by weight aPC; 30% by weight PCZ: 70% by weight aPC; 35% by weight PCZ:65% by weight aPC; 40% by weight PCZ: 60% by weight aPC; 45% by weightPCZ: 55% by weight aPC; and 50% by weight PCZ: 50% by weight aPC; 60% byweight PCZ: 50% by weight aPC; 70% by weight PCZ: 30% by weight aPC; 80%by weight PCZ: 20% by weight aPC; 90% by weight PCZ: 10% by weight aPC;95% by weight PCZ: 5% by weight aPC; 99% by weight PCZ: 1% by weightaPC.

The present formulations are prepared by known procedures usingwell-known and readily available ingredients. Preferably, the PCZ andaPC will be administered parenterally to ensure delivery into thebloodstream in an effective form. Preferably, aPC and PCZ may beformulated according to the disclosure herein. The aPC/PCZ fixedmixtures provide various advantages, including cost savings andadministering convenience and compliance. A vial or other dosagereceptacle of either aPC or PCZ will not have to be opened to obtain asmall quantity of the active ingredient, thus allowing the remainder ofthe active ingredient to become unstable or unusable. The fixed mixtureswill also save time by eliminating mixing and/or multiple dosing. Mixingerrors should also be reduced.

EXAMPLE 3 Formulation of APC/PCZ

A stable lyophilized formulation of aPC is prepared by a process whichcomprises lyophilizing a solution comprising about 2.5 mg/mL aPC or PCZ,about 15 mg/mL sucrose, about 20 mg/mL NaCl, and a sodium citrate bufferhaving a pH greater than 5.5 but less than 6.5. Additionally, the stablelyophilized formulation of aPC and PCZ comprises lyophilizing a solutioncomprising about 5 mg/mL aPC and PCZ, about 30 mg/mL sucrose, about 38mg/mL NaCl, and a citrate buffer having a pH greater than 5.5 and,preferably, less than 6.5.

The ratio of aPC/PCZ:salt:bulking agent (w:w:w) is believed to be animportant factor in a formulation suitable for the freeze dryingprocess. The ratio varies depending on the concentration of aPC/PCZ,salt selection and concentration and bulking agent selection andconcentration. Particularly, a ratio of about 1 part aPC/PCZ to about7.6 parts salt to about 6 parts bulking agent is believed to bepreferred.

A unit dosage formulation of aPC/PCZ suitable for parenteraladministration, preferably subcutaneous administration or continuousintravenous infusion is prepared by mixing aPC, PCZ, NaCl, sucrose, andsodium citrate buffer. After mixing, 4 mL of the solution is transferredto a unit dosage receptacle and lyophilized. The unit dosage receptaclecontaining about 5 mg to about 20 mg of aPC/PCZ, suitable foradministering a dosage of about 0.02 mg/kg/hr to about 0.05 mg/kg/hr topatients in need thereof, is sealed and stored until use.

EXAMPLE 4 aPC/PCZ Formulations

A unit dosage formulation of aPC and PCZ suitable for parenteraladministration is prepared by mixing 0.5 mg of aPC, 1.0 mg of PCZ, 90 mgof NaCl, 100 mg of mannitol, 50 USP units of heparin, 22.5 mg ofaminoacetic acid, and 25 mg of human serum albumin. After mixing, 10 mLof the solution is transferred to a unit dosage receptacle andlyophilized. The unit dosage is sealed.

EXAMPLE 5 aPC/PCZ Formulations

A unit dosage formulation of aPC and PCZ suitable for parenteraladministration administration or continuous infusion is prepared bymixing 1.0 mg of aPC, 1.0 mg of PCZ, 90 mg of NaCl, 100 mg of mannitol,50 USP units of heparin, 22.5 mg of aminoacetic acid, and 25 mg of humanserum albumin. After mixing, 10 mL of the solution is transferred to aunit dosage receptacle and lyophilized. The unit dosage is sealed. Thesame experiment may be repeated where the ratio of aPC: PCZ varies from1 to 99% by weight aPC to 1 to 99% PCZ.

EXAMPLE 6 aPC/PCZ Formulations

A stable lyophilized formulation of aPC/PCZ may be prepared by a processwhich comprises lyophilizing a solution comprising about: 0.125 mg/mL ofaPC and about 2.375 mg of PCZ; or about 0.25 mg/mL of aPC and about 2.25mg of PCZ; or about 0.5 mg/mL of aPC and about 2.0 mg of PCZ; or about0.75 mg/mL of aPC and about 1.75 mg of PCZ; or about 1.0 mg/mL of aPCand about 1.5 mg of PCZ; or about 1.25 mg/mL of aPC and about 1.25 mg ofPCZ; or about 1.5 mg/mL of aPC and about 1.0 mg of PCZ; or about 1.75mg/mL of aPC and about 0.75 mg of PCZ; or about 2.0 mg/mL of aPC andabout 0.5 mg of PCZ; or about 2.25 mg/mL of aPC and about 0.25 mg ofPCZ; or about 2.475 mg PCZ and about 0.25 mg aPC. The aPC and PCZ iscombined with about 15 mg/mL sucrose, about 20 mg/mL NaCl, and a sodiumcitrate buffer having a pH greater than 5.5 but less than 6.5.Additionally, the stable lyophilized formulation of aPC/PCZ compriseslyophilizing a solution comprising about 5 mg/mL of aPC and PCZ (in aratio of about 99% PCZ: 1% aPC and every ratio between 1 % PCZ: 99%aPC), about 30 mg/mL sucrose, about 38 mg/mL NaCl, and a citrate bufferhaving a pH greater than 5.5 but less than 6.5.

Freeze-Dry Microscopy is a useful technique in determining the collapsetemperatures of the frozen solutions that are to be lyophilized. DSC isa useful technique in determining the glass-transition temperature (Tg′)of the frozen solution. The collapse and glass-transition temperaturesare especially helpful in predicting the upper temperature limits thatcan be safely used during the freeze-drying process. Results ofFreeze-Drying Microscopy are complimentary to the glass-transitiontemperature of the Tg′, values obtained by DSC. A collapse temperatureabove −40° C. is optimal for the sample to be processed in aconventional freeze-dryer.

TABLE 1 Freeze dry processing of aPC formulation matrices: FormulationMatrix aPC Sucrose NaCl Collapse Conc. Conc. Conc. Temperature 2.5 mg/mL15 mg/mL  50 mM −59° C. 2.5 mg/mL 15 mg/mL 150 mM −60° C. 2.5 mg/mL 15mg/mL 325 mM −37° C. 5.0 mg/mL 30 mg/mL  50 mM −50° C. to −45° C. 5.0mg/mL 30 mg/mL 150 mM −60° C. to −55° C. 5.0 mg/mL 30 mg/mL 325 mM −64°C. 5.0 mg/mL 30 mg/mL 650 mM −32° C. to −28° C.

The ratio of aPC/PCZ to sucrose to sodium chloride (in 10 or 20 mMcitrate buffer) is believed to be an important formulation variableaffecting the collapse and glass-transition temperatures. To beprocessed in a conventional freeze-dryer, the sodium chlorideconcentration must be high enough (preferably 325 mM for 2.5 mg/mLaPC/PCZ and 650 mM for 5 mg/mL aPC/PCZ formulations) to cause the sodiumchloride to crystallize-out during the freezing part of thefreeze-drying process. Formulations of aPC/PCZ can be processed in aconventional freeze dryer to produce lyophilized products consisting of1 part aPC/PCZ, 6 parts sucrose, and 7.6 parts sodium chloride byweight. One skilled in the art will now appreciate that PCZ may beformulated similarly to the Examples disclosed for aPC. The aPC:PCZratio combined is preferably about 1 part aPC and PCZ, to 6 partssucrose, to about 7.6 parts sodium chloride by weight.

EXAMPLE 7 Stability of aPC in Product Formulations Containing DifferentBulking Agents

Formulations of aPC were prepared to investigate the effect of variousbulking agents on the stability of the molecule. A total of 6 excipientswere added to aPC in phosphate buffer containing no salt. These bulkingagents are glycine, mannitol, sucrose, trehalose, raffinose, andhydroxyethyl starch (HES). The stability of aPC in the phosphate, nosalt, no bulking agent formulation (“control”) was compared to that inthe bulking agent formulations. Samples were stored at 50° C., 40° C.,and 25° C. for various lengths of time. Data from analyses of thesesamples were compared to the initial values (time=0). APTT potency, sizeexclusion-high performance liquid chromatography (SE-HPLC), SDS-PAGE,and protein content assays were used to evaluate the physical andchemical stability of the formulations.

Formulations of aPC were prepared by dissolving aPC in phosphate bufferto 5 mg/mL aPC. Bulking agents were added to portions of the aPCsolution at a ratio of 6:1 (bulking agents to aPC), or 30 mg/mL. Thesamples were lyophilized to 5 mg aPC/vial.

The formulations were put on stability at 50° C. for 14 and 28 days; 40°C. for 28 days, 48 days and 6 months; and 25° C. for 6 and 12 months.For each time point, two vials of each formulation were analyzedindependently as separate samples and data from these samples werecompared to those from initial values (time=0). Analyses included aPCpotency (APTT), SDS-PAGE, percent of aPC monomer, and protein content.

control 25° C. 50° C. 40° C. 6 12 14 28 28 84 6 Via Initia mont monthIniti day day Initi day day mont APTT 1 321 294 236 321 248 248 321 248221 215 (U/mg) 2 321 251 242 321 245 227 321 279 233 176 Monomer 1 99.398.3 96.5 99.3 97.5 97.0 99.3 97.7 96.2 95.1 Content 2 99.2 95.8 96.499.2 97.3 97.1 99.2 97.7 96.1 95.4 glycin 25° C. 50° C. 40° C. 6 12 1428 28 84 6 Via Initi month month Initi day day Initi day day month APTT1 282 233 142 282 164 97 282 191 155 158 (U/mg) 2 321 239 191 321 161142 321 215 152 79 Monomer 1 99.1 98.4 93.3 99.1 97.4 97.2 99.1 97.896.4 95.8 Content 2 99.1 98.4 96.3 99.1 97.3 97.1 99.1 97.7 96.4 95.725° C. mannitol 40° C. 6 12 14 28 28 84 6 Vial Initial month monthInitial day day Initial day day month APTT 1 309 227 255 309 270 245 309273 270 282 (U/mg) 2 321 321 267 321 239 242 321 300 251 191 Monomer 199.2 98.8 97.4 99.2 98. 98.1 99.2 98. 97.6 97.8 Content 2 99.2 98.7 97.699.2 98. 98.0 99.2 98. 97.6 97.8 25° C. Suc 40° C. 6 12 14 28 28 84 6Vial Initial month month Initial day day Initial day day month APTT 1327 300 288 327 300 288 327 267 306 285 (U/mg) 2 297 300 306 297 291 291297 321 242 294 Monomer 1 99.2 99.0 98.5 99.2 98. 98.9 99.2 98. 98.598.9 Content 2 99.2 99.0 98.5 99.2 98. 98.9 99.2 98. 98.5 98.9 Trehalo25° C. 50° C. 40° C. 6 12 14 28 28 84 6 Via Initi month month Initi dayday Initi day day month APTT 1 312 291 282 312 258 282 312 273 276 276(U/mg) 2 309 315 282 309 270 215 309 303 245 255 Monomer 1 99.2 99.098.4 99.2 98.6 98.8 99.2 98.8 98.4 98.7 Content 2 99.2 98.8 98.4 99.298.6 98.8 99.2 98.7 98.4 98.7 raffinose 25° C. 50° C. 40° C. 6 12 14 2828 84 6 Via Initi month month Initi day day Initi day day month APTT 1321 270 255 321 261 258 321 276 273 279 (U/mg) 2 288 285 306 288 255 264288 270 239 255 Monomer 1 99.1 99.0 97.0 99.1 98.6 98.7 99.1 98.7 98.498.6 Content 2 99.1 99.0 98.2 99.1 98.6 98.7 99.1 98.7 98.4 98.6 HES 25°C. 50° C. 40° C. 6 12 14 28 28 84 6 Via Initi month month Initi day dayIniti day day month APTT 1 282 188 176 282 182 164 282 194 185 145(U/mg) 2 285 245 215 285 188 161 285 176 152 103 Monomer 1 97.8 95.692.2 97.8 93.0 91.8 97.8 93.7 90.6 88.7 Content 2 97.8 95.3 91.8 97.892.9 91.0 97.8 92.9 90.5 88.5

There were no significant changes in pH, color, package characteristicsand physical appearance for any of the samples over the one yearstability time period. When analyzed by the APTT and SE-HPLC procedures,the HES and glycine formulation had less physical stability (throughaggregation) and chemical stability (potency) when compared to thecontrol. The mannitol formulation offered slightly better physical andchemical stability than the control, and the remaining formulations,sucrose, trehalose and raffinose, all demonstrated even more superiorphysical and chemical stability when compared to the control. Therefore,mannitol, sucrose, trehalose and raffinose, as bulking agents in aPCformulations, offer increased chemical and physical stability whencompared to an aPC formulation without a bulking agent or those havingglycine or HES. Albumin or a similar known pharmaceutical excipients maybe added, for example, to improve stability.

EXAMPLE 8 Stability of Recombinant Human APC

Two lots of a lyophilized formulation of recombinant human aPC werestored for 1 month at 40° C./75% relative humidity, and then analyzedfor possible degradation. The stability of aPC was also monitored afterreconstitution with sterile water and storage for up to 72 hours atambient temperature. The lyophilized aPC product consisted of 10 mg aPC,60 mg sucrose, 76 mg sodium chloride, and 15.1 mg citrate per vial. TheaPC in this formulation is stable in the dry state for at least 1 monthwhen stored at 40° C./75% relative humidity, and in solution for 24hours when stored at ambient temperature.

Both lots were prepared using the same unit formula of 10 mg aPC, 60 mgsucrose, 76 mg sodium chloride, and 15.1 mg citrate per vial. Bothlyophilized lots of aPC were stored for 1 month at 40° C./75% relativehumidity and the stability of aPC was monitored using the APTT potencyassay, ion-pairing HPLC for quantitation of aPC peptides and massspectrometry for quantitation of protein variant forms. One lot was alsoreconstituted with sterile water, to 1 mg/mL aPC, and held at ambienttemperature. The stability of aPC in solution was monitored at the 0, 1,4, 8, 24, 48 and 72 hour time points using the APTT and massspectrometry methods.

There was no loss of aPC activity and an insignificant amount ofstructural degradation of the molecule after storage in the dry statefor one month at 40° C./75% relative humidity. The aPC in thisformulation is stable for up to 24 hours at about 1 mg/mL to about 4mg/mL after reconstitution.

1. A method of treating a human patient with a hypercoagulable state orprotein C deficiency which comprises administering to said patientactivated Protein C (aPC) and protein C zymogen (PCZ).
 2. The methodaccording to claim 1 wherein said hypercoagulable state or protein Cdeficiency is associated with a disease or condition selected from:sepsis, severe sepsis, septic shock, disseminated intravascularcoagulation, purpura fulminans, major trauma, undergoing or recoveringfrom surgery, burns, adult respiratory distress syndrome, bone marrowand other organ transplantations, deep vein thrombosis, heparin-inducedthrombocytopenia, sickle cell disease, thalassemia, viral hemorrhagicfever, thrombotic thrombocytopenic purpura, hemolytic uremic syndrome,unstable angina, myocardial infarction, meningococcemia, melioidosis,complications during pregnancy, preeclampsia, eclampsia, amniotic fluidembolism, placental abruption, and chemotherapy.
 3. The method of claim2 wherein the hypercoagulable state or protein C deficiency is selectedfrom sepsis, severe sepsis, and septic shock.
 4. The method of any ofclaims 1 to 3 wherein the activated Protein C is administered to thepatient first, followed by administration of protein C zymogen.
 5. Themethod of any of claims 1 to 3 wherein the protein C zymogen isadministered to the patient first, followed by administration ofactivated Protein C.
 6. The method of any of claims 1 to 3 wherein theactivated Protein C and protein C zymogen are administeredsimultaneously to the patient.
 7. The method of any of claims 1 to 3wherein the administration of the activated Protein C and protein Czymogen is alternated back and forth between the activated Protein C andthe protein C zymogen.
 8. The method of any of claims 1 to 3 wherein theactivated Protein C is administered by continuous infusion at a dose ofabout 1 μg/kg/hr to about 50 μg/kg/hr.
 9. The method of any of claims 1to 3 wherein the activated Protein C is administered by continuousinfusion at a dose of about 5 μg/kg/hr to about 30 μg/kg/hr.
 10. Themethod of any of claims 1 to 3 wherein the activated Protein C isadministered by continuous infusion at a dose of about 5 μg/kg/hr toabout 25 μg/kg/hr.
 11. The method of any of claims 1 to 3 wherein theactivated Protein C and the protein C zymogen are administered toachieve an aPC plasma range of about 20 ng/ml to about 160 ng/ml. 12.The method of claim 11 wherein the activated Protein C plasma range isabout 25 ng/ml to about 100 ng/ml.
 13. The method of claim 8, whereinthe dose of protein C zymogen is about 60 to about 120 μg/kg/day with orwithout a bolus of about 100 to about 500 μg/kg.
 14. The method of claim13, wherein the dose of PCZ is about 90 μg/kg/day with or without abolus of about 100 to about 500 μg/kg.
 15. The method of claim 2 orclaim 3, wherein the aPC and/or the PCZ are produced recombinantly. 16.The method of claim 2 or claim 3, wherein the aPC and/or the PCZ areplasma derived.
 17. A pharmaceutical formulation which comprisesactivated Protein C, protein C zymogen, and a pharmaceutical carrierwherein the weight:weight ratio of the activated Protein C and theprotein C zymogen is selected from: 5% by weight protein C zymogen: 95%by weight activated Protein C; 10% by weight protein C zymogen: 90% byweight activated Protein C; 15% by weight protein C zymogen: 85% byweight activated Protein C; 20% by weight protein C zymogen: 80% byweight activated Protein C; 25% by weight protein C zymogen: 75% byweight activated Protein C; 30% by weight protein C zymogen: 70% byweight activated Protein C; 35% by weight protein C zymogen: 65% byweight activated Protein C; 40% by weight protein C zymogen: 60% byweight activated Protein C; 45% by weight protein C zymogen: 55% byweight activated Protein C; and 50% by weight protein C zymogen: 50% byweight activated Protein C; 60% by weight protein C zymogen: 40% byweight activated Protein C; 70% by weight protein C zymogen: 30% byweight activated Protein C; 80% by weight protein C zymogen: 20% byweight activated Protein C; 90% by weight protein C zymogen: 10% byweight activated Protein C; 95% by weight protein C zymogen: 5% byweight activated Protein C; and 99% by weight protein C zymogen: 1% byweight activated Protein C.
 18. The formulation of claim 17 wherein theratio is: 5% by weight protein C zymogen: 95% by weight activatedProtein C.
 19. The formulation of claim 17 wherein the ratio is: 10% byweight protein C zymogen: 90% by weight activated Protein C.
 20. Theformulation of claim 17 wherein the ratio is: 15% by weight protein Czymogen: 85% by weight activated Protein C.
 21. The formulation of claim17 wherein the ratio is: 20% by weight protein C zymogen: 80% by weightactivated Protein C.
 22. The formulation of claim 17 wherein the ratiois: 25% by weight protein C zymogen: 75% by weight activated Protein C.23. The formulation of claim 17 wherein the ratio is: 30% by weightprotein C zymogen: 70% by weight activated Protein C.
 24. Theformulation of claim 17 wherein the ratio is: 35% by weight protein Czymogen: 65% by weight activated Protein C.
 25. The formulation of claim17 wherein the ratio is: 40% by weight protein C zymogen: 60% by weightactivated Protein C.
 26. The formulation of claim 17 wherein the ratiois: 45% by weight protein C zymogen: 55% by weight activated Protein C.27. The formulation of claim 17 wherein the ratio is: 50% by weightprotein C zymogen: 50% by weight activated Protein C.
 28. Theformulation of claim 17 wherein the ratio is: 55% by weight protein Czymogen: 45% by weight activated Protein C.
 29. The formulation of claim17 wherein the ratio is: 60% by weight protein C zymogen: 40% by weightactivated Protein C.
 30. The formulation of claim 17 wherein the ratiois: 65% by weight protein C zymogen: 35% by weight activated Protein C.31. The formulation of claim 17 wherein the ratio is: 70% by weightprotein C zymogen: 30% by weight activated Protein C.
 32. Theformulation of claim 17 wherein the ratio is: 75% by weight protein Czymogen: 25% by weight activated Protein C.
 33. The formulation of claim17 wherein the ratio is: 80% by weight protein C zymogen: 20% by weightactivated Protein C.
 34. The formulation of claim 17 wherein the ratiois: 85% by weight protein C zymogen: 15% by weight activated Protein C.35. The formulation of claim 17 wherein the ratio is: 90% by weightprotein C zymogen: 10% by weight activated Protein C.
 36. Theformulation of claim 17 wherein the ratio is: 95% by weight protein Czymogen: 5% by weight activated Protein C.
 37. The formulation of claim17 wherein the ratio is: 99% by weight protein C zymogen: 1% by weightactivated Protein C.